The long-term goal of Project 1 is to define the mechanisms responsible for the ryanodine receptor (RyR1)[unreadable] myopathies, malignant hyperthermia and central core disease (MH/CCD). Our immediate objectives are to[unreadable] create suitable models for human disease in mice and use them to study how mutations of RyR1 alter[unreadable] intracellular Ca2+ homeostasis and to create a noninvasive diagnostic test for MH that has a high degree of[unreadable] specificity and sensitivity.[unreadable] HYPOTHESIS I: Heterozygous MH "knock-in" mice model Human MH susceptibility.[unreadable] A 1.1. To create a "hot-spot" region 2 MH 'knock in' mouse line (RyR1 G2434R). In addition to our recently[unreadable] created R163C (region 1) and T4826I (region 3) RyR1 MHS mice. A 1.2.To determine the relationship between[unreadable] the clinical MH phenotype and myoplasmic resting free calcium ([Ca2+],) in vivo, with contracture properties[unreadable] in vitro for each heterozygous MH/CCD mouse. A1.3 To determine the relationship between IVCT and[unreadable] myoplasmic resting [Ca2+] in myotubes from human biopsy samples. A1.4 To determine if genetic[unreadable] background can rescue the birth lethal phenotype seen in homozygous MH/CCD mice. A1.5 To determine if[unreadable] genetic background will lower or raise [Ca2+]i or sensitivity to halothane in heterozygous MH/CCD mice.[unreadable] HYPOTHESIS II: Mutations responsible for human MH/CCD increase passive RyR1 "leak" and alter[unreadable] the dynamics of EC coupling by enhancing ECCE and SOCE.[unreadable] A 2.1 To analyze heterozygous and homozygous MH/CCD myotubes for abnormalities in EC coupling and[unreadable] two forms of Ca2+ entry, ECCE and SOCE. A 2.2 To establish how halothane and dantrolene enhance and[unreadable] diminish aberrant Ca2+ signaling in MH/CCD myotubes. A 2.3 To validate murine MH/CCD myotube model[unreadable] results with myotubes obtained from humans with an analogous mutation.[unreadable] Hypothesis III: The "Funnel" approach can be used to create a non-invasive screening test for[unreadable] MH that has both a high degree of specificity and a high degree of sensitivity.[unreadable] A 3.1. We will analyze whole blood transcriptional profiles from patients with known MHS RyR1 mutations[unreadable] and use the Funnel approach to select a set of predictive markers to be used for future MHS screening.[unreadable]